Recombinant Human Proprotein Convertase 7/PCSK7 Protein, CF Summary
Leu38-Thr667, with a 10-His tag between Arg141 and Ser142
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CF stands for Carrier Free (CF). We typically add Bovine Serum Albumin (BSA) as a carrier protein to our recombinant proteins. Adding a carrier protein enhances protein stability, increases shelf-life, and allows the recombinant protein to be stored at a more dilute concentration. The carrier free version does not contain BSA.
In general, we advise purchasing the recombinant protein with BSA for use in cell or tissue culture, or as an ELISA standard. In contrast, the carrier free protein is recommended for applications, in which the presence of BSA could interfere.
|Formulation||Supplied as a 0.2 μm filtered solution in Tris, NaCl, CaCl2 and Glycerol.|
|Shipping||The product is shipped with polar packs. Upon receipt, store it immediately at the temperature recommended below.|
|Stability & Storage:||Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
- Activation Buffer: 50 mM Tris, 10 mM CaCl2, 0.15 M NaCl, 0.05% Brij 35, pH 7.5 (TCNB)
- Assay Buffer: 25 mM Tris, pH 7.0
- Recombinant Human Proprotein Convertase 7/PCSK7 (PCSK7) (Catalog # 2984-SE)
- Bacterial Thermolysin (Thermolysin) (Catalog # 3097-ZN)
- 1,10 Phenanthroline (Sigma, Catalog # 320056), 0.6 M stock in DMSO
- Substrate: L-PyroGlu-Arg-Thr-Lys-Arg-AMC (Catalog # ES013)
- F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
- Fluorescence Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent
- Activate rhPCSK7 by diluting to 50 µg/mL in Activation Buffer containing 2.0 µg/mL Thermolysin.
- Incubate activation reaction at 37 °C for 4 hours.
- Add 1,10 Phenanthroline at a final concentration of 10 mM to stop Thermolysin activity.
- Dilute Substrate to 200 µM in Assay Buffer.
- Diluted activated rhPCSK7 to 2.0 µg/mL in Assay Buffer.
- In a plate load 50 µL of 2.0 µg/mL rhPCSK7 to wells and include a Substrate Blank of 50 µL of Assay Buffer.
- Start the reaction by adding 50 µL of 200 µM Substrate to wells.
- Read at excitation and emission wavelengths of 380 nm and 460 nm (top read), respectively, in kinetic mode for 5 minutes.
- Calculate specific activity:
Specific Activity (pmol/min/µg) =
|Adjusted Vmax* (RFU/min) x Conversion Factor** (pmol/RFU)|
|amount of enzyme (µg)|
*Adjusted for Substrate Blank
**Derived using calibration standard 7-amino, 4-Methyl Coumarin (Sigma, Catalog # A-9891).
- rhPCSK7: 0.1 µg
- Substrate: 100 µM
Background: Proprotein Convertase 7/PCSK7
The human PCSK7 gene encodes proprotein convertase 1 (PC7), which is also known as PC8 and lymphoma PC (1, 2). As a serine protease of the furin/PC family, the deduced amino acid sequence of human PC7 consists of a signal peptide (residues 1 to 37), a propeptide (residue 38 to 141), and a mature chain (residues 142 to 785) that consists of extracellular (residues 142 to 667), transmembrane (residues 668 to 688) and cytoplasmic (residues 689 to 785) domains. Autocatalytic processing results in the removal of the propeptide, which confers potent inhibitory activity toward the enzyme (3-5). The purified recombinant human PCSK7 corresponds to the extracellular domain of the mature chain with a 10X His tag at the N-terminus. However, it may also contain the propeptide, as suggested by the N-terminal sequencing results. Thermolysin treatment increases its activity, which may be due to degradation of the propeptide by thermolysin.
- Bruzzaniti, A. et al. (1996) Biochem. J. 314:727.
- Meerabux, J. et al. (1996) Cancer Res. 56:448.
- Van de Loo, J.-W.H.P. et al. (1997) J. Biol. Chem. 272:27116.
- Bhattacharjya, S. et al. (2000) Biochemistry 39:2868.
- Seidah, N.G. and M. Chretien (2004) in Handbook of Proteolytic Enzymes (ed. Barrett, et al.) pp. 1877, Academic Press, San Diego.
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